1. Field of the Invention
The present invention provides improved methods and reagents for the detection of Hepatitis C virus, a causative agent of non-A, non-B hepatitis. The reagents include oligonucleotide primers for both reverse transcription of the viral RNA genome and subsequent amplification by the polymerase chain reaction of the cDNA so produced. Sequence specific oligonucleotide probes are provided for the detection of amplified HCV nucleic acid sequences. The primers and probes are useful in a diagnostic test for the detection of HCV infection. This diagnostic test has important applications in both clinical and epidemiological settings.
2. Description of Related Art
Hepatitis C virus (HCV) is one of an unknown number of agents responsible for non-A, non-B hepatitis (NANBH). The prototypical HCV was identified from a eDNA clone of a blood-borne NANBH virus obtained from the plasma of an infected chimpanzee as reported in Choo et al., 1989, Science 244:359-362. Nucleotide sequences of genes from this prototype HCV are described in European Patent Publication Nos. 318,216: 388,232; and 398,748. The nucleotide sequence of the HCV genome was reported and compared to related virus in Choo et al., 1991, Proc. Natl. Acad. Sci. USA 88:2451-2455.
Sequences from other strains have since been reported. The genome of HCV exhibits a large degree of nucleic acid sequence heterogeneity between isolates. The isolation of cDNA from the HCV RNA genome was reported in Kubo et al., 1989, Nuc. Acids Res. 17: 10367-10372. The authors constructed a reverse transcription primer based on the sequence reported in Choo et al., 1989, supra. The fragment of the HCV genome isolated showed 79.8% homology with the prototype HCV sequence. Obtained by a similar protocol, sequences from three different regions were reported in Takeuchi et al., 1990, Gene 91:287-291. Sequence homology with the prototype sequence was reported as low as 73.5% for one of the regions.
The HCV genomic sequence from a strain isolated from Japanese patients was reported in Kato et al., 1990, Proc. Natl. Acad. Sci. USA 87:9524-9528. The sequence showed 77.4% homology with the prototype HCV genome over the region compared. A sequence for the entire coding region of the HCV genome reported in Takamizawa et al., 1991, J. Virol. 65 (3):1105-1113 showed 77% homology with the prototype HCV strain.
In Japan, two main strains, designated strains K1 and K2, have been observed based on a 400 nucleotide region of the genome. These strains have sequence homology with the prototype HCV sequence as low as 67% (see Enomoto et al., 1990, Biochem. Biophys. Res. Commun. 170(3):1021-1025). The K2 strain could be further subdivided into two groups, designated Ka and Kb, with about 20% nucleotide variation between the groups and about 5% nucleotide variation within each group.
Similar levels of homology with the prototype HCV sequence were reported in Kato et al., 1990, J. Clin. Invest. 86:1764-1767. From 15 patients, cDNA segments were amplified and sequenced. The amplified portion, 37 nucleotides corresponding to positions 3525-3561 in the prototype sequence, showed 68-78% homology with the prototype HCV sequence.
HCV genomic RNA can be detected in sera by creating cDNA from the genomic RNA, amplifying the cDNA with the polymerase chain reaction, and subsequently probing with sequence-specific oligonucleotides. Because of the sequence heterogeneity among HCV strains, primers and probes are likely to be strain-specific, unless a region in which the sequence is conserved across strains can be found. One such conserved region is at the 5'-end of the HCV genome.
The 5'-terminal noncoding sequence of the HCV genome was first reported in Okamoto et al., 1990, Japan J. Exp. Med. 60(3):167-177. Comparison between two strains suggested that the 5'-terminal noncoding sequence is conserved. The conserved nature of the 5'-terminal noncoding sequence of the HCV genome was also reported in Hah et al., 1991, Proc. Natl. Acad. Sci. USA 88:1711-1715. Partial sequences of a 341 nucleotide region obtained from 11 HCV isolates collected from individuals from five continents were compared. Seven sequences showed complete homology with the prototype sequence; the remaining four showed between one and five base mismatches.
In Okamoto et al., 1990, Japan J. Exp. Med. 60(3):215-222, primers for various regions of the HCV genome, including the conserved 5' noncoding region were described. The primers selected from conserved regions successfully amplified nucleic acid from most of the strains tested; primers chosen from heterogeneous regions amplified nucleic acid from a smaller subset of strains. However, the amplification efficiency with these primers was low. The reference describes a two-stage PCR amplification; the second round of amplification was performed on the previously amplified target region using a second set of primers nested within the region amplified by the first set of primers.
A PCR amplification requiring two rounds of amplification using two sets of primers was also reported in Garson et al., 1990, Lancet 335:1419-1422. A region encoding a nonstructural protein (NSS) was amplified. Due to sequence heterogeneity, there are HCV sequences not recognized by these primers (see Garson et al., 1990, Lancet 336:878-879). Consequently, primers in the conserved 5' noncoding region were tried, but to obtain sufficient sensitivity, a two-sage amplification using sets of nested primers was still necessary. Amplification of the 5'-terminal region using a two-stage amplification with nested primers was also reported in Kanai et al., 1990, Lancet 336:245.
Amplification using two rounds of amplification with nested primers is not only inefficient but also greatly increases the probability of contamination. The problems of contamination are well known in the art; opening the reaction tube to change primers and add reagents between amplification steps is best avoided if at all possible. The contamination problem is further aggravated by the need to change reaction conditions between the initial reverse transcription step and the subsequent PCR amplification.
There is still a need for primer oligonucleotides for amplifying HCV sequences, each chosen from a conserved region so that all, or almost all, strains will be amplified, and amplification methods efficient enough that amplification with one set of primers is sufficient. There is also a need for probe oligonucleotides for the detection of the amplified eDNA chosen from a conserved region in between the two conserved regions to which the primers hybridize. Reaction protocol and reagents are needed that allow reverse transcription and PCR to occur using the same reagents, thereby eliminating the need to open the reaction tube during the amplification process.
Moreover, ten percent of NANBH cases are nonreactive with the prototype capsid and envelope antigens. Chaudhary et al., 1991 J. Clinical Microbiology 29:2329-2330 and Hosein et al., 1991, PNAS 8:3647-3651. Thus, the development of PCR based diagnostics and antigens encoded by new isolates will improve the dependability of serologically based diagnostic tests. The present invention meets these needs by providing primers, probes, and methods for detecting HCV.